The primary focus of our funded research is to determine the regulatory mechanisms that govern and maintain meiotic commitment using budding yeast as a model organism. An exogenous signal induces cells to sequentially enter meiosis and then differentiate into gametes. Our studies seek to determine how cells integrate external signals with intracellular regulatory networks to commit to meiosis or to maintain meiotic commitment if the signal is removed or replaced by a mitosis-inducing signal. Our main goals are to identify the cell-cycle regulators that govern meiotic commitment, determine how the regulators function within the network, and determine how checkpoint mechanisms monitor meiotic commitment. Our results will uncover general mechanisms of cell-cycle regulation in both mitosis and meiosis. An essential tool for our studies is the use of time-lapse microscopy. We developed a microfluidics assay to manipulate cells? external signals as we are monitoring meiotic commitment in individual cells with time-lapse microscopy. This assay is a powerful tool to allow us to dissect the cell-cycle regulatory mechanisms of meiotic commitment. By studying individual cells, we can more precisely identify cells that fail to maintain meiotic commitment in the presence of mitosis-inducing signals. And, we can identify perturbations in meiotic duration in different mutant backgrounds. In this supplement proposal, I am requesting funds for the replacement of my aging microscope system, with a Nikon eclipse Ti2. Our system has served us well, but is no longer reliable and is likely near the end of its lifetime. The Nikon eclipse Ti2 will allow us to perform the time-lapse imaging for the experiments proposed.